1. Field of the Invention
The present invention generally relates to a process for a mass-propagation and regeneration of plants from protoplasts of shoot primordia. More specifically, it relates to a process for a mass-production of woody plants through shoot primordia, and a process for a regeneration of plants from protoplasts of shoot primordia.
2. Description of the Related Art
The propagation process for woody plants is classified into two fundamental processes, i.e., sexual reproduction, and asexual reproduction, including cutting and tissue culture. In the sexual reproduction of allogamous plants, the traits of parent plants are not necessarily transmitted to their progeny, due to pollen variations. In the cases of interspecific hybrid and F.sub.1 hybrid generated by hybrid vigor as well as polyploid plant, the genotype of parent plant cannot be transmitted to its progeny without change.
The asexual reproduction includes cutting, which has been extensively used for a long time as a propagation method for excellent plants. However, cutting is not appropriate for the propagation of woody plants, due to a lower propagation rate, lower rooting ability, the necessity of a field for production of plant pieces for cutting, and a limited season for cutting. On the other hand, the tissue culture methods have recently been rapidly developed. There are disadvantages in that a mass-propagation of progeny plants having the same traits as the parent plant is difficult because chromosomal aberration and gene mutation occur at a high frequency during the regeneration of shoots from calli. Moreover, in many cases, the long-term subculture of callus lowers its differentiation potency.
More specifically, for broad-leaved trees such as poplar, eucalypt and the like, there have been attempts at mass-propagation using culture of various kinds of organs, such as shoot tip, axillary bud, cotyledon, hypocotyl, stem etc. However, when starting from the shoot tip, since once a callus is induced from the shoot tip and the callus is then redifferentiated into shoots, the same problem occurs. On the other hand, in the case of direct induction of adventitious shoot from stem or the like, i.e., micropropagation, continuously to obtain shoots, the stems or the like must be planted at appropriate intervals, resulting in a disadvantage in commercial mass-propagation.
Moreover, for gymnosperms such as conifer, it is possible to generate an embryoid. For example, Mostafa M. et al. (U.S. Pat. No. 4,217,730 issued on Aug. 19, 1980) prepared embryoid from Pseudotsuga menziesii by suspension culture. In this method, although the regeneration-term is shorter than in conventional tissue culture (organogenesis), a rate of the conversion of the embryoid into an entire plant is as low as 15 to 50%. Another disadvantage of this method is that, since young cotyledons are used, a large amount of seeds is necessary. Therefore, this method does not form a seed-less mass-propagation process for excellent individuals.
Accordingly, in the conventional tissue culture, a genetically stable and rapid mass-propagation technique for woody plants has not been accomplished.
Recently, for the propagation of annual plants other than woody plants, a "shoot primordium" method was presented (Ryuso Tanaka et al., Jpn. J. Genet. Vol. 58, 65-70, 1983; Japanese Unexamined Patent Publication No. 59-132822; and Japanese Unexamined Patent Publication No. 59-132823).
This shoot primordium method is a method using shoot tip. In this method, shoot tips are removed from an annual plant, and the shoot tips are rotary-cultured in an artificial medium under a particular temperature, illumination, and rotation rate to form globular cell aggregates containing shoot primordia, which are then cultured for shooting to obtain for a short term a large amount of young plants stably maintaining the genetic traits of their parent.
The present inventors found that the "shoot primordium" method, which has already been applied to annual plants such as water-melon, maize, rice, morning glory, Swertia, Papaver and the like, is also applicable to perennial woody plants (Japanese Unexamined Patent Publication No. 62-55020).
Shoot primordium is a propagation body, and by propagating the shoot primordia a large number of clones can be produced from a parent plant via the shoot primordia.
In the shoot primordium method, so far shoot primordia are stationary-cultured for shooting on a solid medium such as agar medium. In this stationary-culture, however, the shooting ratio is as low as about 30%.
Since a protoplast has no cell wall, it is capable of cell fusion or useful genes can be introduced therein. If plants can be regenerated from a protoplast thus manipulated, it is possible to create novel plants, for example, plants providing a higher yield and better taste, and exhibiting a greater resistance to pest and bad weather conditions, and so on. In herbaceous plants, many kinds of species are capable of regeneration from protoplasts. However, for woody plants, a regeneration of protoplast into plant is successful only in limited kinds of plants, such as "Trovita" orange (Kobayashi, S. et al., Jpn. J. Breeding, Vol. 34, supplement 2, 32-33, 1984), paper mulberry (Oka, S. and K. Oyama, Jpn. J. Breeding, Vol. 34, supplement 2, 26-27, 1984; Japanese Unexamined Patent Publication No. 61-192283), poplar (Ito, K. et al. the present inventors, Japanese Patent Application No. 61-64800; Russell, J.A. and B.H. McCown, Plant Sci., 46, 133-142, 1986), Sandalwood (Rao, R.S. and P. Ozias-Akins, Protoplasma, 124, 80-86, 1985) and Ulmus (STICKLEN, M.B. et al., Plant Sci., 47, 29-34, 1986).
A reason for this may be that isolated protoplasts excrete substances such as polyphenols into a medium during culturing, and the excreted substances have an adverse affect on the survival and cell division, resulting in an inhibition of the regeneration of the plant.
To overcome this obstacle, recently, principally for monocotyledon, an immature embryo or an inflorescence is used to form an embryogenic callus, and from the callus, protoplasts are prepared and cultured to regenerate plants. However, this method is disadvantageous in that the preparation of a starting material is time-consuming, the method can be carried out only in a limited season, and it is difficult to provide stable materials due to genetic mutation occurring during the callus formation.
Therefore, it is desirable to develop those materials which are genetically stable, have differentiation ability, and can propagate in a large amount.